Multinuclear magnetic resonance studies on serine protease transition state analogues

Abstract31P Nuclear Magnetic Resonance (NMR) studies were performed on mono‐ and diisopropylphosphoryl derivatives of α‐chymotrypsin, trypsin, and subtilisin. Questions addressed included the pKa of the active center Asp…His…Ser traid in both species. While the pKa in the diisopropylphosphoryl derivatives is near 7.4 (found in this and other laboratories earlier) and reflects a nearly normal imidazolium titration curve, the apparent pKa in the monoisopropylphosphoryl enzymes (obtained by “aging” of the diisopropylphosphoryl derivatives and monitored by 31P NMR) is between 9.7 and 11.4 depending on the protease. This latter “titration” of the 31P NMR signal is reversible and presumably reflects the interaction of the imidazolium positive charge with the monoanionic phosphodiester. Of the two tetrahedral intermediates, the properties of the monoisopropylphosphoryl enzyme are probably more representative of the tetrahedral oxyanionic intermediate invoked during peptide hydrolysis. The same NMR technique was used to determine the action of PAM (pyridine‐2‐aldoxime methiodide, a known “antidote” for acetylcholinesterase inactivated by diisopropylfluorophosphate), on the inactivated enzy